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In order to address the objective of this research, we isolated bacterial strains from L. uliginosus growing under field conditions in an arsenic contaminated site in the central region of Portugal. In this context, 22 isolates were obtained from root nodules of L. uliginosus. All isolates were slow growers, showed little congo red absorption, and had an alkaline reaction on BTB, indicated by a blue colour, which is usually produced by Bradyrhizobium spp.[30] (Supplemental Fig. S2). Congo red is often incorporated in culture media for isolating rhizobia or for testing the purity of rhizobia cultures. Rhizobia typically do not absorb congo red or absorb it weakly, while other bacteria absorb it strongly.
The assessment of genotypic diversity of the natural population nodulating L. uliginosus was achieved by ERIC-PCR. The analysis of the fingerprinting patterns of each isolate showed the existence of several clusters (Fig. 1). This dendrogram was used to determine the similarities among isolates. Results showed the presence of a high genetic diversity in the population, despite the high contamination by As in the original soil. These isolates showed multiple fingerprinting patterns and no single dominant genotype was apparent from our results.
Figure 1.
Dendrogram showing the diversity of root nodule bacteria (Bradyrhizobium sp.) isolated from L. uliginosus, derived from ERIC-PCR fingerprints using UPGMA method, at 85% similarity.
Arsenic tolerance
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Results obtained for As tolerance after 72 h (Fig. 2, Supplemental Fig. S3 and Table 1) showed that, for the highest concentration tested (200 mg of As mL−1), most isolates were considered As sensitive (percentage of growth inhibition (GI) of 80%−100%). Only two isolates, 13 and 12, were considered as moderately tolerant (GI 60%−80%) and tolerant (GI < 60%), respectively. On the other hand, for 50 mg·mL−1 of As about 36% of isolates (isolates 8, 12, 13, 14, 15, 16, 17 and 23) were tolerant, and 18% were considered moderately tolerant (isolates 7, 18, 19 and 22). However, an inverse situation was verified for 10 and 5 mg·mL−1 of As, where no isolates were found sensitive. While for 10 mg·mL−1 64% of the isolates were tolerant, for the lowest concentration of As used, 5 mg·mL−1, a large majority of isolates (95%) was considered as tolerant.
Figure 2.
Tolerance of the L. uliginosus isolates to different As concentrations (5, 10, 50 and 200 mg·mL−1). Isolates were classified according to the percentage of growth inhibition relative to controls grown in the absence of As, being considered sensitive (80%−100%), moderately tolerant (60%−80%) and tolerant (< 60%). Stacked-columns indicate the percentage of isolates in the three tolerance classes for each As concentration.
Table 1. Nodulation phenotype of L. uliginosus plants inoculated with the different isolates upon different As concentrations.
Isolates As concentration (mg·mL−1) 0 0.5 5 10 20 3 w 6 w 3 w 6 w 3 w 6 w 3 w 6 w 3 w 6 w 8 + + + + + + + + − − + + + + + + + + − − + + + + + + + + − − 10 + + − ± − ± − − − − + + − ± − ± − − − − + + − ± − ± − − − − 12 + + + + + + ± ± − − + + + + + + ± ± − − + + + + + + ± ± − − 15 + + − + − + + + − + + + + + + + + + − + + + + + + + + + − + 21 + + − + + + − + − + + + + + + + − + − − + + + + + + − + − − 23 + + − + − + − + − + + + − + − + + + − + + + + + + + + + − − 24 + + + + − + − + − − + + + + − + − + − − + + − − − + − + − − w, incubation weeks; +, presence of nodules; −, absence of nodules; ±, presence of small and white nodules. Effects of arsenic on the symbiosis
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For the evaluation of the effects of As on the symbiosis with L. uliginosus, seven isolates (namely 8, 10, 12, 15, 21, 23 and 24) belonging to different clusters (Fig. 1) and with different levels of tolerance to As (Supplemental Fig. S3) were chosen to inoculate L. uliginosus cv. Sunrise seedlings. Assays were performed with six As concentrations (0.5, 5, 10, 20, 100 and 200 mg of As mL−1) and results were recorded three and six weeks after the addition of bacterial inocula and As to the seedlings. Briefly, plants did not tolerate the highest As concentrations used, 100 and 200 mg·mL−1, and died one week after As addition. Arsenic toxicity affected the symbiosis and the different isolates also showed different symbiotic performances as the arsenic concentration increased up to 20 mg·mL−1 (Fig. 3).
Figure 3.
Effect of As on the symbiosis. Values represent the average of the index of effectiveness (Es) ± SD of L. uliginosus plants inoculated with the bacterial isolates and grown with different As concentrations (0, 0.5, 5, 10 and 20 mg·mL−1). Different letters express significant differences between plants inoculated with each isolate at several As concentrations according to Tukey's HSD test at at P < 0.05.
One of the effects of As in the symbiosis was demonstrated by the delay in nodulation (Table 1), especially for higher concentrations of As tested, when compared to the controls without the addition of As. A more drastic effect was the impairment of the symbiosis by the lack of nodulation. For the highest As concentration allowing plant survival, 20 mg·mL−1, only plants inoculated with isolates 15, 21 and 23 presented pink root nodules (i.e., functional) at least in one of the replicates. In this case, nodulation was delayed, but the symbiosis remained effective in some replicates, depending on the isolate tested. Plants inoculated with isolates 8 and 12 did not show delayed nodulation but nodules were only formed at As concentrations up to 10 mg·mL−1. However, at this concentration plants inoculated with isolate 12 had small and white nodules (i.e., ineffective). Nodulation was also observed (with the presence of pink nodules) in plants inoculated with isolate 24 until 10 mg of As mL−1 but with a delay of 3 weeks. Plants inoculated with isolate 10 and growing with 0.5 and 5 mg of As mL−1 showed also a delay in nodulation and nodules were small and white. This nodulation phenotype indicates an ineffective symbiosis, inversely to what happened with plants grown without the addition of As which had pink nodules.
These plant-inoculation experiments using several As concentrations were also performed to evaluate the symbiotic effectiveness, by determining the shoot dry weight of L. uliginosus plants after 6 weeks of growth. The As concentrations used in these experiments were higher than those usually present in groundwater used for irrigation and in soils in various countries, e.g. 3,10 µg·L−1 and 22 mg·kg−1, respectively, in Argentina, or 3,700 µg·L−1 and 196 mg·kg−1, respectively, in India[43]. All the isolates chosen for these experiments were considered effective in the absence of As and the respective indices of effectiveness (Es), under these conditions, ranged between 35% and 55% (Fig. 3). These isolates also showed different levels of symbiotic performance and indices of effectiveness upon different As concentrations. In general, results were congruent with the observed nodulation phenotypes (Table 1). Isolates 15, 21 and 23 were able to establish an efficient symbiosis with the host plant at 20 mg of As mL−1. Plants inoculated with isolate 15 showed an increase in the aerial part and the consequent increase in the indices of effectiveness at the concentration of 0.5 mg As mL−1, which was near 75% and the highest Es of these experiments. This strain was therefore considered highly effective in the presence of As (0.5 mg·mL−1). However, for the other two isolates, 21 and 23 respectively, no significant differences were found between the various treatments, including the control without As addition, and their respective indices of effectiveness were lower but still considered as effective in nitrogen fixation. On the other hand, isolates 8, and 24, were able to establish an efficient symbiosis until the concentration of 10 mg of As mL−1. For the first isolate, no significant differences were found between the various treatments (0.5, 5 and 10 mg As mL−1) including the control without addition of As. The indices of effectiveness of plants inoculated with isolate 12 and with 0.5 and 5 mg of As mL−1 did not show significant differences between each other and the control (without As addition). However, for 10 mg·mL−1 of As the index of effectiveness was very low (19%) and the symbiosis was considered as ineffective, the plants presenting small and white nodules as previously mentioned. Lastly, plants inoculated with isolate 10 had a very weak performance in the presence of As (with plants having small and white nodules) and were only able to establish an efficient symbiosis when As was completely absent, showing indices of effectiveness significantly different from all the remaining treatments.
Interestingly, among the isolates that were able to establish an efficient symbiosis while sustaining the highest As concentration tolerated by plants, 20 mg·mL−1 (isolates 15, 21 and 23), isolate 15 was also among those with higher indices of effectiveness in the absence of As. Moreover, this isolate had the highest Es shown, near 75% at 0.5 mg·mL−1 of As being considered as highly effective in nitrogen fixation.
16S rRNA region phylogeny
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Aligned sequences of the partial 16S rRNA region were used to construct the phylogenetic tree shown in Fig. 4. The isolates from this study were all clustered with Bradyrhizobium spp. Sequences obtained from isolates 8, 10, 15, 23 and 24 shared 100% sequence identity and the closest strains were Bradyrhizobium spp. isolated from Lotus uliginosus (Bradyrhizobium sp. 3LBC, 8LBI, SEMIA 839 and NZP2309[21,28]), Cytisus triflorus (Bradyrhizobium sp. CTS12), Cytisus scoparius (Bradyrhizobium genosp. AD Cs6020[44]) Vigna unguiculata L. (Bradyrhizobium sp. VUPMI37) and Ulex europaeus (Bradyrhizobium sp. ICMP 12674[45]). Isolates 12 and 21 shared 100% sequence identity and the closest strains were Bradyrhizobium spp. isolated from Glycine max (Bradyrhizobium japonicum GI-4 and J5 and Bradyrhizobium sp. 323S2[46,47]), Pigeonpea (Bradyrhizobium sp. RP6) and Erythrina brucei (Bradyrhizobium shewense ERR11T [48]).
Figure 4.
Phylogeny of the partial 16S rRNA gene with a total of 1189 aligned positions. Confidence bootstrap values are presented near each node. NCBI GenBank accession codes are presented next to each strain. Isolates obtained in this work are in bold. Rhizobium leguminosarum USDA 2370T (U29386) was selected as an outgroup.
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The main impact of this work is the possible use of autochthonous legume plants and their micro-symbionts, such as the symbioses with L. uliginosus and Bradyrhizobium, for the phytostabilization of contaminated soils, helping its fertilization. Results reveal that root nodule bacteria isolated from L. uliginosus growing in polluted soils, mainly those tolerant to the highest concentration of arsenic, can be symbiotically effective upon high As concentrations. This can be a very interesting aspect and can be used in the future, in bioremediation experiments on contaminated soils using native legumes, since it could have a positive ecological impact in those sites. We consider that the dual function of As bioremediation plus soil nitrogen enhancement can be achieved by effective symbiotic nodulation in affected As-metal-soils. Such a tolerant and functional symbiosis can support vegetation cover, stabilizing As-contaminated soils, and consequently it could be a smart practice for phytostabilization.
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About this article
Cite this article
Soares R, Fareleira P, Colavolpe B, Ruiz OA, Videira e Castro I. 2023. Root nodule bacteria isolated from Lotus uliginosus for future use in phytostabilization of arsenic contaminated soils. Grass Research 3:8 doi: 10.48130/GR-2023-0008
Root nodule bacteria isolated from Lotus uliginosus for future use in phytostabilization of arsenic contaminated soils
- Received: 12 January 2023
- Accepted: 20 April 2023
- Published online: 10 May 2023
Abstract: In recent decades there has been growing concern around heavy metals and metalloid contamination in soil. Arsenic (As) is a ubiquitous trace metalloid. The high levels of this metalloid in soils are a consequence of human activities and also from natural inputs. In general, the biodiversity of microorganisms and plants decreases drastically in contaminated soils. The knowledge that some leguminous plants, mainly certain species of Lotus, are growing well in such soils has attracted our attention for studying symbioses that are well adapted to harsh conditions. In this work we studied the rhizobial population existing in the root nodules of native Lotus uliginosus Sch. growing in a central region of Portugal. This legume grows in soils particularly affected by As due the discharge of industrial liquid effluents from fertilizer and chemical facilities. Diversity and tolerance to different concentrations of As of root nodule bacteria were studied. Our results showed that the symbioses between L. uliginosus and As tolerant Bradyrhizobium isolates were efficient when a nutrient medium containing high As concentrations was used. The present work highlights the capacity of L. uliginosus to grow and establish nitrogen-fixing symbioses in soils strongly contaminated with As and its potential for future use to promote vegetation cover to stabilize As contaminated soils.